Hrp labeled goat anti rabbit antibody

Total proteins from the hippocampus tissue were extracted using a total proteins Extraction Kit (BestBio; cat. no. BB‐3101‐100T). The protein samples were firstly separated in a 10% SDS‐PAGE gel and then transferred to PVDF membranes. Blocked with 5% non‐fat milk, the membranes were incubated with the primary antibodies overnight at 4°C: Pink1 (1:1000, Abcam; cat. no. ab23707), Parkin (1:1000, Abcam; cat. no. ab233434), LC3B (1:1000, Abcam; cat. no. ab63817), SQSTM1/p62 (1:1000, Cell Signaling Technology; cat. no. 23214), caspase‐1 (1:1000, Abcam; cat. no. ab1872), IL‐1β (1:1000, Cell Signaling Technology; cat. no. 12703), IL‐1R1 (1:1000, Santa Cruz Biotechnology; cat. no. sc‐689), NF‐κB (1:1000, Cell Signaling Technology; cat. no. 8242), pNF‐κB (1:1000, Cell Signaling Technology; cat. no. 3033), TNF‐α (1:1000, Abcam; cat. no. ab205587), and iNOS (1:1000, Abcam; cat. no. ab15323). The next day, the secondary antibody HRP‐labeled goat anti‐rabbit antibody (1:1000, Cell Signaling Technology; cat. no. 7074) was added to incubate the membranes at 4°C for 2 h. The immunoblots were visualized by an imaging densitometer (ImageQuant LAS 500, GE Healthcare Bio‐Sciences AB,). The band intensity was quantified through the software ImageJ. β‐actin was used as the control.

In detail, cells were collected and lysed in radio immunoprecipitation assay (RIPA) buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholine, and 1 mM NaF), and the protein concentration was measured using a BCA protein assay kit (Pierce Biotechnology). Proteins were separated on 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After 2 h, the membranes were blocked with 5% skim milk and incubated with the primary antibodies at 4 °C overnight. After that, the membranes were washed with TBST for three times and incubated with HRP-labeled goat anti-rabbit antibody [Cell Signaling Technology (CST), USA] for 0.5 h. Finally, the immunoreactive signals were detected using the ECL detection system (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific, IL, USA). The following primary antibodies were applied: rabbit anti-HIF1α (36169S, CST, USA), rabbit anti-P-p65 (3033S, CST, USA), rabbit anti-GPR116 (ab136262, Abcam, Shanghai, China), rabbit anti-p65 (8242S, CST, USA), rabbit anti-β-actin (4970 L, CST, USA), rabbit anti-GNA11 (ab153951, abcam, UK).